Chimeric proteins

ABSTRACT

Chimeric proteins possessing Kunitz-type domain 1 of TFPI-2 and Kunitz-type domain 2 of TFPI are disclosed, as are muteins of TFPI and TFPI-2. Nucleic acid sequences, expression vectors and transformed host cells encoding and capable of producing the disclosed chimeric proteins and muteins are also disclosed. Finally, methods for prevention and treatment of septic shock using the chimeric proteins and muteins are disclosed.

This application is a continuation of application Ser. No. 08/256,521,filed Aug. 5, 1994, U.S. Pat. No. 5,589,357.

BACKGROUND OF THE INVENTION

This invention relates to chimeric proteins capable of simultaneouslybinding and inhibiting factor VIIa/tissue factor complex (factor VIIa/TFcomplex) and factor Xa, expression vectors coding for the proteins ofthe invention, host cells transformed with the expression vectors,methods for producing the proteins, pharmaceutical compositionscontaining the proteins, methods of treatment or prevention of septicshock using the proteins, methods of inhibiting coagulation disordersand monoclonal antibodies against the proteins.

Human Tissue Factor Pathway Inhibitor (TFPI) is a plasma proteaseinhibitor. Based on homology study, TFPI appears to be a member of theKunitz-type basic protease inhibitor gene superfamily. TFPI functions inat least two ways: 1) Inhibition of the catalytic activity of factorVIIa/TF complex and 2) By binding to the active site of factor Xa. Theprimary sequence of TFPI, deduced from its cDNA sequence, indicates thatthe protein contains three Kunitz-type domains. The first of these,Kunitz-type domain 1, is believed to be required for the efficientbinding to and inhibition of factor VIIa/TF complex, which is enhancedby the presence of the second Kunitz-type domain, Kunitz-type domain 2.Kunitz-type domain 2 is required for the efficient binding to andinhibition of factor Xa by TFPI. The function of the third Kunitz-typedomain, Kunitz-type domain 3, is unknown. TFPI has no known enzymaticactivity and probably inhibits the activity of protease targets in astoichiometric manner, namely, binding of one Kunitz-type domain to theactive site of one protease molecule. TFPI is also known as LipoproteinAssociated Coagulation Inhibitor (LACI), tissue factor inhibitor (TFI)and extrinsic pathway inhibitor (EPI).

Mature TFPI is a polypeptide of about 276 amino acids in length with anegatively charged amino terminal end and a positively charged carboxylterminal end. The C-terminal tail (i.e., the sequence following the lastcysteine residue of Kunitz-type domain 3) is highly basic and isbelieved to aid in the localization of TFPI to cell surfaces by bindingto glycosaminoglycan (including heparin) or phospholipids found on cellsurfaces. This cell surface localization property is believed to beimportant for full anticoagulant activity and for optimal inhibition offactor Xa TFPI contains 18 cysteine residues and forms 9 disulfidebridges when correctly folded. The primary sequence contains threeAsn-X-Ser/Thr N-linked glycosylation consensus sites with asparagineresidues at positions 145, 196, and 256. The carbohydrate component ofmature TFPI is approximately 30% of the mass of the protein. Data fromproteolytic mapping and mass spectral data imply that the nativecarbohydrate moieties are heterogeneous. Native TFPI is also found to bephosphorylated at the serine residue at position 2 of the protein tovarying degrees. The role of phosphorylation at Ser-2 in TFPI functionhas yet to be elucidated.

Recently, another protein with a high degree of structural andfunctional similarity to TFPI has been identified, as described inSprecher et al. Proc. Natl. Acad Sci. USA 91:3353-3357 (1994). Thepredicted secondary structure of this 213 amino acid residue protein,called TFPI-2, is virtually identical to TFPI having three Kunitz-typedomains, 9 disulfide bridges, an acidic amino terminus and a basiccarboxy terminus. The three Kunitz-type domains of TFPI-2 exhibit 43%,35% and 53% primary sequence identity with TFPI Kunitz-type domains 1,2, and 3, respectively. Compared with TFPI, recombinant TFPI-2 stronglyinhibits the amidolytic activity of factor VIIa/TF complex and weaklyinhibits factor Xa activity. TFPI-2 is reported to bind with greateraffinity to factor VIIa/TF complex than does TFPI, whereas TFPI binds tofactor Xa with greater affinity than does TFPI-2.

The presumed P 1-reactive site in Kunitz-type domain 1 of TFPI-2 isarginine, as contrasted with lysine in TFPI. The P1-reactive site inKunitz-domain 2 of TFPI-2 is glutamate, as contrasted with arginine inTFPI. Also, the Kunitz-type domain 2 of TFPI-2 contains two additionalamino acid residues between the fourth and fifth cysteine residues. Thespacer region between Kunitz-type domain 1 and 2 in TFPI-2 is muchshorter than the corresponding TFPI spacer region. One or more of thesedifferences may result in the different affinities of the two proteinsfor factor VIIa/TF complex and Xa.

TFPI has been shown to prevent mortality in a lethal Escherichia coil(E. coli) septic shock baboon model. Creasey et al, J. Clin. Invest.91:2850-2860 (1993). Administration of TFPI at 6 mg/kg body weightshortly after infusion of a lethal dose of E. coli resulted in survivalin all five TFPI-treated animals with significant improvement in qualityof life, compared with a mean survival time for the five control animalsof 39.9 hours. The administration of TFPI also resulted in significantattenuation of the coagulation response, of various measures of cellinjury and significant reduction in pathology normally observed in E.coli sepsis target organs, including kidneys, adrenal glands, and lungs.Due to its clot-inhibiting properties, TFPI may also be used to preventproblems associated with thrombosis and clotting such as duringmicrovascular surgery. For example, U.S. Pat. No. 5,276,015 disclosesthe use of TFPI in a method for reducing thrombogenicity ofmicrovascular anastomoses wherein TFPI is administered at the site ofthe microvascular anastomoses contemporaneously with microvascularreconstruction.

TFPI has been isolated from human plasma and from human tissue culturecells, including HepG2, Chang liver and SK hepatoma cells. RecombinantTFPI has been expressed in mouse C127 cells, baby hamster kidney cells,Chinese hamster ovary cells and human SK hepatoma cells. RecombinantTFPI from the mouse C127 cells has been shown in animal models toinhibit tissue-factor induced coagulation. A non-glycosylated form ofrecombinant TFPI has also been produced and isolated from Escherichiacoli (E. coli) cells as disclosed in U.S. Pat. No. 5,212,091. This formof TFPI has been shown to be active in the inhibition of bovine factorXa and in the inhibition of human tissue factor-induced coagulation inplasma. In some assays, the E. coli-produced TFPI has been shown to bemore active than TFPI derived from SK hepatoma cells. Methods have beendisclosed for purification of recombinant TFPI from yeast cell culturemedium, such as in Petersen et al, J. Biol. Chem. 18:13344-13351 (1993).Truncated forms of recombinant TFPI have also been studied, as describedin Hamamoto et al. J. Biol. Chem. 268:8704-8710 (1993) and Petersen etal. ibid.

Petersen et al. ibid have attempted to produce TFPI and variants ofTFPI, including: 1) variants of TFPI in which the complete C-terminalone third of the polypeptide, including Kunitz-type domain 3, wasdeleted; 2) variants of TFPI in which just Kunitz-type domain 3 wasdeleted; and 3) variants of TFPI in which the basic portion of thepeptide, C-terminal to Kunitz-type domain 3, were deleted. They foundthat high yields were obtained only with the first variant. This variantwas heterogeneously glycosylated, and its anti-coagulant activity was5-50 fold lower than full-length TFPI obtained from mammalian cells.

A need exists, therefore, for a method to produce in high yield, aprotein molecule that possesses at least the equivalent, if notenhanced, anticoagulant and other activities, of TFPI and that hasreduced glycosylated moieties that would in turn result in reducedimmunogenicity of the protein upon administration to a mammal.

SUMMARY OF THE INVENTION

It is, therefore, one of the objects of the present invention to providea protein that possesses equivalent or enhanced anticoagulant activity,in particular, factor VIIa/TF complex and/or factor Xa inhibitoryactivity, as compared to full length TFPI obtained from mammalian cells,yeast cells or bacterial cells.

Another object of the present invention is to produce in high yield, aprotein having factor VIIa/TF complex and/or factor Xa inhibitoryactivity.

Another object of the present invention is to provide a protein havingthe activity of TFPI, TFPI-2, or both which target cell surfaces as wellor better than TFPI or TFPI-2.

It is further one of the objects of the present invention to provide aprotein that has reduced glycosylated moieties as compared to thevariants or full length TFPI described in Petersen et al, ibid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of a replicable cloning vehicle (designatedpLACI 4.1) including a DNA sequence coding for the TFPI protein.

FIG. 2A shows a Coomassie stained gel containing full length andtruncated versions of TFPI initially expressed as ubiquitin and α-factorfusions. FIG. 2B shows the results of a Western blot of the gel of FIG.2A with antisera specific for the amino-terminal end of TFPI. For both2A and 2B, the lanes were loaded as follows: soluble protein isolatedfrom culture medium in which control cells (V2) were grown (Lane 1);soluble protein isolated from yeast cells expressing a LACI fragment(amino acids 1-161) (Lane 2); soluble protein isolated from yeast cellsexpressing full length LACI (Lane 3); protein isolated from controlyeast cells (Lane 4); protein isolated from yeast cells expressing aLACI fragment (amino acids 1-161) (Lane 5); protein isolated from yeastcells expressing full length LACI (Lane 6); and molecular weight markers(Lane 7).

FIG. 3 A shows a Coomassie stained gel containing full length andtruncated versions of TFPI before and after digestion with N glycanaseto remove the sugars. FIG. 3B shows the results of a Western blot of thegel of FIG. 3A with antisera specific for the amino-terminal end ofTFPI. The gel was loaded as follows: proteins from V2 control cells(Lane 1); proteins from yeast cells expressing a LACI fragment (aminoacids 1-161) (Lane 2); proteins from yeast cells expressing full lengthLACI (Lane 3); proteins from V2 control cells treated with N-glycanase(Lane 4); proteins from yeast cells expressing a LACI fragment (aminoacids 1-161) treated with N-glycanase (Lane 5); proteins from yeastcells expressing full length LACI treated with N-glycanse (Lane 6); LACIstandard (Lane 7) and molecular weight markers (Lane 8).

FIG. 4 shows the results of an amidolytic assay performed on truncatedand full-length TFPI initially expressed as α-factor fusions andsecreted from the yeast cell.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "TFPI" refers to the coagulation inhibitorTissue Factor Pathway Inhibitor, also known as Lipoprotein AssociatedCoagulation Inhibitor (LACI), Tissue Factor Inhibitor (TFI) andExtrinsic Pathway Inhibitor (EPI). The nucleotide sequence encoding TFPIand the predicted amino acid sequence of TFPI have been disclosed inU.S. Pat. No. 4,966,852, which is herein incorporated by reference.

As used herein, the term "TFPI-2" refers to a coagulation inhibitor, thenucleotide sequence and predicted amino acid sequence of which have beenreported by Sprecher et al, Proc. Nat. Acad. Sci. USA (1994)91:3353-3357. The disclosure of Sprecher et al is herein incorporated byreference.

As used herein, the term "factor VIIa/TF/Xa binding protein" refers toproteins capable of binding to the factor VIIa/TF complex therebyinhibiting the function of the complex and further capable of bindingfactor Xa thereby inhibiting its function. The factor VIIa/TF/Xa bindingproteins contain one or more Kunitz-type domains derived from TFPI (ormuteins thereof) and one or more Kunitz-type domains from derived TFPI-2(or muteins thereof).

As used herein, the term "first Kunitz-type domain" refers to aminoacids

    __________________________________________________________________________    Cys                                                                              Ala                                                                              Phe                                                                              Lys                                                                              Ala                                                                              Asp                                                                              Asp                                                                              Gly                                                                              Pro                                                                              Cys                                                                              Lys                                                                              Ala                                                                              Ile                                                                              Met                                                                              Lys                                                                              Arg                              Phe                                                                              Phe                                                                              Phe                                                                              Asn                                                                              Ile                                                                              Phe                                                                              Thr                                                                              Arg                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Glu                                                                              Phe                                                                              Ile                                                                              Tyr                                                                              Gly                              Gly                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Asn                                                                              Gln                                                                              Asn                                                                              Arg                                                                              Phe                                                                              Glu                                                                              Ser                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                              Lys                                                                              Met                                                                              Cys                                                                               SEQ ID NO: 1!                                                       __________________________________________________________________________

of TFPI and the amino acid sequence

    __________________________________________________________________________    Cys                                                                              Leu                                                                              Leu                                                                              Pro                                                                              Leu                                                                              Asp                                                                              Tyr                                                                              Gly                                                                              Pro                                                                              Cys                                                                              Arg                                                                              Ala                                                                              Leu                                                                              Leu                                                                              Leu                                                                              Arg                              Tyr                                                                              Tyr                                                                              Tyr                                                                              Asp                                                                              Arg                                                                              Tyr                                                                              Thr                                                                              Gln                                                                              Ser                                                                              Cys                                                                              Arg                                                                              Gln                                                                              Phe                                                                              Leu                                                                              Tyr                                                                              Gly                              Gly                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Asn                                                                              Ala                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Tyr                                                                              Thr                                                                              Trp                                                                              Glu                                                                              Ala                                                                              Cys                                                                              Asp                              Asp                                                                              Ala                                                                              Cys                                                                               SEQ ID NO: 2!                                                       __________________________________________________________________________

of TFPI-2; the term "second Kunitz-type domain" refers to amino acids

    __________________________________________________________________________    Cys                                                                              Phe                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Pro                                                                              Gly                                                                              Ile                                                                              Cys                                                                              Arg                                                                              Gly                                                                              Tyr                                                                              Ile                                                                              Thr                                                                              Arg                              Tyr                                                                              Phe                                                                              Tyr                                                                              Asn                                                                              Asn                                                                              Gln                                                                              Thr                                                                              Lys                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Arg                                                                              Phe                                                                              Lys                                                                              Tyr                                                                              Gly                              Gly                                                                              Cys                                                                              Leu                                                                              Gly                                                                              Asn                                                                              Met                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Glu                                                                              Thr                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                              Asn                                                                              Ile                                                                              Cys                                                                               SEQ ID NO: 3!                                                       __________________________________________________________________________

of TFPI and amino acids

    __________________________________________________________________________    Cys                                                                              Arg                                                                              Leu                                                                              Gln                                                                              Val                                                                              Ser                                                                              Val                                                                              Asp                                                                              Asp                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Ser                                                                              Thr                                                                              Glu                              Lys                                                                              Tyr                                                                              Phe                                                                              Phe                                                                              Asn                                                                              Leu                                                                              Ser                                                                              Ser                                                                              Met                                                                              Thr                                                                              Cys                                                                              Glu                                                                              Lys                                                                              Phe                                                                              Phe                                                                              Ser                              Gly                                                                              Gly                                                                              Cys                                                                              His                                                                              Arg                                                                              Asn                                                                              Arg                                                                              Ile                                                                              Glu                                                                              Asn                                                                              Arg                                                                              Phe                                                                              Pro                                                                              Asp                                                                              Glu                                                                              Ala                              Thr                                                                              Cys                                                                              Met                                                                              Gly                                                                              Phe                                                                              Cys                                                                               SEQ ID NO: 4!                                              __________________________________________________________________________

of TFPI-2; and the term "third Kunitz-type domain" refers to amino acids

    __________________________________________________________________________    Cys                                                                              Leu                                                                              Thr                                                                              Pro                                                                              Ala                                                                              Asp                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Arg                                                                              Ala                                                                              Asn                                                                              Glu                                                                              Asn                                                                              Arg                              Phe                                                                              Tyr                                                                              Tyr                                                                              Asn                                                                              Ser                                                                              Val                                                                              Ile                                                                              Gly                                                                              Lys                                                                              Cys                                                                              Arg                                                                              Pro                                                                              Phe                                                                              Lys                                                                              Tyr                                                                              Ser                              Gly                                                                              Cys                                                                              Gly                                                                              Gly                                                                              Asn                                                                              Glu                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Thr                                                                              Ser                                                                              Lys                                                                              Gln                                                                              Glu                                                                              Cys                                                                              Leu                              Arg                                                                              Ala                                                                              Cys                                                                               SEQ ID NO: 5!                                                       __________________________________________________________________________

of TFPI and amino acids

    __________________________________________________________________________    Cys                                                                              Tyr                                                                              Ser                                                                              Pro                                                                              Lys                                                                              Asp                                                                              Glu                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Ser                                                                              Ala                                                                              Asn                                                                              Val                                                                              Thr                                                                              Arg                              Tyr                                                                              Tyr                                                                              Phe                                                                              Asn                                                                              Pro                                                                              Arg                                                                              Tyr                                                                              Arg                                                                              Thr                                                                              Cys                                                                              Asp                                                                              Ala                                                                              Phe                                                                              Thr                                                                              Tyr                                                                              Thr                              Gly                                                                              Cys                                                                              Gly                                                                              Gly                                                                              Asn                                                                              Asp                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Val                                                                              Ser                                                                              Arg                                                                              Glu                                                                              Asp                                                                              Cys                                                                              Lys                              Arg                                                                              Ala                                                                              Cys                                                                               SEQ ID NO: 6!                                                       __________________________________________________________________________

of TFPI-2.

As used herein, the term "C-terminal tail" refers to the amino acidsequences which are carboxy-terminal to the third Kunitz-type domain ofTFPI or of TFPI-2, i.e.,

    __________________________________________________________________________    Lys                                                                              Lys                                                                              Gly                                                                              Phe                                                                              Ile                                                                              Gln                                                                              Arg                                                                              Ile                                                                              Ser                                                                              Lys                                                                              Gly                                                                              Gly                                                                              Leu                                                                              Ile                                                                              Lys                                                                              Thr                              Lys                                                                              Arg                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Lys                                                                              Gln                                                                              Arg                                                                              Val                                                                              Lys                                                                              Ile                                                                              Ala                                                                              Tyr                                                                              Glu                                                                              Glu                                                                              Ile                              Phe                                                                              Val                                                                              Lys                                                                              Asn                                                                              Met                                                                               SEQ ID NO: 7!                                                 __________________________________________________________________________

for TFPI and

    __________________________________________________________________________    Ala                                                                              Lys                                                                              Ala                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Lys                                                                              Lys                                                                              Lys                                                                              Met                                                                              Pro                                                                              Lys                                                                              Leu                                                                              Arg                                                                              Phe                                                                              Ala                              Ser                                                                              Arg                                                                              Ile                                                                              Arg                                                                              Lys                                                                              Ile                                                                              Arg                                                                              Lys                                                                              Lys                                                                              Gln                                                                              Phe                                                                               SEQ ID NO: 8!                               __________________________________________________________________________

for TFPI-2. These sequences are highly basic and may be involved in cellsurface localization by glycosaminoglycan (including heparin) orphospholipid binding. Further description and explanation of Kunitz-typedomains 1, 2 and 3 and the P₁ -reactive site for each domain may befound in Girard et al, Nature, 338:518-520 (1989).

As used herein, the term "P₁ -reactive site" refers to the active sitecleft of a Kunitz-type domain. Alteration of the amino acid residuepresent in the P₁ position can profoundly alter the binding, andtherefore the inhibitory effect, of the Kunitz-type domain to its targetprotease.

As used herein, the term "chimeric protein" refers to a polypeptideconsisting of one or more domains from different proteins or mutationswithin a single protein giving the characteristics of another protein.For example, a chimeric protein as used herein would include a factorVIIa/TF/Xa binding protein containing SEQ ID NO: 2 and SEQ ID NO: 1.

As used herein, the term "mutein" refers to a normal or wild-typesequence in which 1-5 amino acid substitutions have been made. Forexample, a mutein in Kunitz-type domain 1 of TFPI SEQ ID NO: 1! may bemade in the P₁ position by changing a lysine residue to an arginineresidue. This substitution has the effect of altering the properties ofKunitz-type domain 1 of TFPI, including affinity for factor VIIa/TFcomplex, to those of Kunitz-type domain 1 of TFPI-2.

As used herein, the term "pharmaceutically acceptable carrier" refers toa medium which does not interfere with the effectiveness of thebiological activity of the active ingredient and which is not toxic tothe hosts to which it is administered.

As used herein, the term "pharmacologically effective amount" refers tothe amount of protein administered to the host that results in reductionof morbidity and mortality resulting from the condition being treated.Conditions that may be treated include sepsis, septic shock andthrombosis disorders, including thrombosis during and after microsurgeryand thrombosis from abrupt reclosure after angioplasty. The exact amountadministered depends on condition, severity, subject etc., but may bedetermined by routine methods. The term "pharmacologically effectiveamount" also refers to the amount of protein administered to the hostthat prevents morbidity and mortality resulting associated with acondition. Conditions that may be prevented include sepsis, septic shockand thrombosis disorders, including thrombosis during and aftermicrosurgery and thrombosis from abrupt reclosure after angioplasty. Theexact amount administered depends on condition, severity, subject etc.,but may be determined by routine methods.

Factor VIIa/TF/Xa binding proteins of the invention include muteins ofTFPI and TFPI-2, the muteins having single or multiple amino acidsubstitutions. Muteins within the scope of this definition include: (a)TFPI or TFPI-2 muteins having 1-5 conservative amino acid substitutionsthat do not substantially change the conformation of the molecule; (b)TFPI or TFPI-2 muteins with amino acid substitutions that eliminate oneor more of the three sites for N-linked glycosylation; (c) TFPI muteinshaving 1-5 amino acid substitutions that change a residue of TFPI to acorresponding residue of TFPI-2; (d) TFPI-2 muteins having 1-5 aminoacid substitutions that change a residue of TFPI-2 to a correspondingresidue of TFPI; (e) TFPI or TFPI-2 muteins with amino acidsubstitutions in P₁ reactive sites in one or more Kunitz-type domains;and (f) TFPI or TFPI-2 muteins with amino acid substitutions atpositions within 5 amino acids of the P₁ reactive sites in one or moreKunitz-type domains. In a preferred embodiment, the lysine residue inthe P₁ -reactive site of the first Kunitz-type domain of TFPI SEQ ID NO:1! is replaced with arginine. The mutein has the following sequence:

    __________________________________________________________________________    Asp                                                                              Ser                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Glu                                                                              Glu                                                                              His                                                                              Thr                                                                              Ile                                                                              Ile                                                                              Thr                                                                              Asp                                                                              Thr                                                                              Glu                                                                              Leu                              Pro                                                                              Pro                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Met                                                                              His                                                                              Ser                                                                              Phe                                                                              Cys                                                                              Ala                                                                              Phe                                                                              Lys                                                                              Ala                                                                              Asp                                                                              Asp                              Gly                                                                              Pro                                                                              Cys                                                                              Arg                                                                              Ala                                                                              Ile                                                                              Met                                                                              Lys                                                                              Arg                                                                              Phe                                                                              Phe                                                                              Phe                                                                              Asn                                                                              Ile                                                                              Phe                                                                              Thr                              Arg                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Glu                                                                              Phe                                                                              Ile                                                                              Tyr                                                                              Gly                                                                              Gly                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Asn                                                                              Gln                                                                              Asn                              Arg                                                                              Phe                                                                              Glu                                                                              Ser                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                                                                              Lys                                                                              Met                                                                              Cys                                                                              Thr                                                                              Arg                                                                              Asp                                                                              Asn                              Ala                                                                              Asn                                                                              Arg                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Thr                                                                              Thr                                                                              Leu                                                                              Gln                                                                              Gln                                                                              Glu                                                                              Lys                                                                              Pro                                                                              Asp                                                                              Phe                              Cys                                                                              Phe                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Pro                                                                              Gly                                                                              Ile                                                                              Cys                                                                              Arg                                                                              Gly                                                                              Tyr                                                                              Ile                                                                              Thr                                                                              Arg                              Tyr                                                                              Phe                                                                              Tyr                                                                              Asn                                                                              Asn                                                                              Gln                                                                              Thr                                                                              Lys                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Arg                                                                              Phe                                                                              Lys                                                                              Tyr                                                                              Gly                              Gly                                                                              Cys                                                                              Leu                                                                              Gly                                                                              Asn                                                                              Met                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Glu                                                                              Thr                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                              Asn                                                                              Ile                                                                              Cys                                                                              Glu                                                                              Asp                                                                              Gly                                                                              Pro                                                                              Asn                                                                              Gly                                                                              Phe                                                                              Gln                                                                              Val                                                                              Asp                                                                              Asn                                                                              Tyr                                                                              Gly                              Thr                                                                              Gln                                                                              Leu                                                                              Asn                                                                              Ala                                                                              Val                                                                              Asn                                                                              Asn                                                                              Ser                                                                              Leu                                                                              Thr                                                                              Pro                                                                              Gln                                                                              Ser                                                                              Thr                                                                              Lys                              Val                                                                              Pro                                                                              Ser                                                                              Leu                                                                              Phe                                                                              Glu                                                                              Phe                                                                              His                                                                              Gly                                                                              Pro                                                                              Ser                                                                              Trp                                                                              Cys                                                                              Leu                                                                              Thr                                                                              Pro                              Ala                                                                              Asp                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Cys                                                                              Arg                                                                              Ala                                                                              Asn                                                                              Glu                                                                              Asn                                                                              Arg                                                                              Phe                                                                              Tyr                                                                              Tyr                                                                              Asn                              Ser                                                                              Val                                                                              Ile                                                                              Gly                                                                              Lys                                                                              Cys                                                                              Arg                                                                              Pro                                                                              Phe                                                                              Lys                                                                              Tyr                                                                              Ser                                                                              Gly                                                                              Cys                                                                              Gly                                                                              Gly                              Asn                                                                              Glu                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Thr                                                                              Ser                                                                              Lys                                                                              Gln                                                                              Glu                                                                              Cys                                                                              Leu                                                                              Arg                                                                              Ala                                                                              Cys                                                                              Lys                              Lys                                                                              Gly                                                                              Phe                                                                              Ile                                                                              Gln                                                                              Arg                                                                              Ile                                                                              Ser                                                                              Lys                                                                              Gly                                                                              Gly                                                                              Leu                                                                              Ile                                                                              Lys                                                                              Thr                                                                              Lys                              Arg                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Lys                                                                              Gln                                                                              Arg                                                                              Val                                                                              Lys                                                                              Ile                                                                              Ala                                                                              Tyr                                                                              Glu                                                                              Glu                                                                              Ile                                                                              Phe                              Val                                                                              Lys                                                                              Asn                                                                              Met.                                                                              SEQ ID NO: 9!.                                                   __________________________________________________________________________

Muteins of TFPI and TFPI2 containing one or more amino acidsubstitutions may be prepared by appropriate mutagenesis of the sequenceof a recombinant cloning vehicle encoding TFPI or TFPI-2, usingtechniques known to those skilled in the art. Techniques for mutagenesisinclude, without limitation, site specific mutagenesis. Site specificmutagenesis can be carried out using any number of procedures known inthe art. These techniques are described by Smith, Annual Review ofGenetics, 19:423 (1985), and modifications of some of the techniques aredescribed in Methods in Enzymology, 154, part E, (eds.) Wu and Grossman(1987), chapters 17, 18, 19, and 20. A preferred procedure when usingsite specific mutagenesis is a modification of the Gapped Duplex sitedirected mutagenesis method. The general procedure is described byKramer, et al., in chapter 17 of the Methods in Enzymology, above.Another technique for generating point mutations in a nucleic acidsequence is the use of PCR techniques, including overlapping PCR, asdescribed in PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, (eds.)Innis, Gelfand, Sninsky and White (Academic Press, 1990).

The muteins of TFPI and TFPI-2 may also be truncated at the end of thesecond Kunitz-type domain. Such truncated molecules retain the abilityto bind factor VIIa/TF complex and Xa yet can be expressed at higherlevels in such organisms as yeast. The truncated TFPI and TFPI-2 muteinswill likely lead to enhanced recovery of a product containing correctlyfolded Kunitz-type domains due to the removal of six cysteine residuesin the third Kunitz-type domain. The truncated muteins may also have thetail sequence of TFPI or of TFPI-2 attached at the carboxy-terminal endto give the mutein cell surface-binding ability, preferably by bindingto glycosaminoglycans (including heparin) or phospholipid at the cellsurface.

Chimeric proteins capable of binding factor VIIa/TF and factor X_(a) andcontaining various portions of TFPI or TFPI-2 are also within the scopeof the invention. One class of proteins within the scope of theinvention can be represented by the following generic formula:

    A-(X.sub.1).sub.a -B-(X.sub.2).sub.b -C

wherein A and C are optional flanking peptides, the flanking peptidesindependently containing 1-100 amino acids;

wherein B is an optional spacer peptide, the spacer peptide containing1-25 amino acids;

wherein each X₁ is -D-K₁ -E-

where D,E are peptides of 1-25 amino acids,

where K₁ is independently Kunitz-type domain 1 from TFPI or TFPI-2 SEQID NO: 1 or SEQ ID NO: 2! or a mutein of the aforementioned Kunitz-typedomains;

wherein each X₂ is -F-K₂ -G-

where F,G are peptides of 1-25 amino acids,

where K₂ is independently Kunitz-type domain 2 from TFPI or TFPI-2 SEQID

NO: 3 or SEQ ID NO: 4! or a mutein of the aforementioned Kunitz-typedomains;

wherein a,b are integers from 0-6; and

wherein the molecule is not native TFPI or TFPI-2.

A,B,C,D,E,F,G may independently comprise portions of native TFPI orTFPI-2 sequences. For example, B, D, E, F, and G may independentlycomprise peptide sequences between Kunitz-type domain 1 and 2 of TFPI orTFPI-2, or peptide sequences between Kunitz-type domains 2 and 3 of TFPIand TFPI-2. The flanking peptides A and C may also have cell surfacelocalization properties and may be the C-terminal tail sequence fromTFPI or TFPI-2 SEQ ID NO: 7 or SEQ ID NO: 8!. Alternatively, other cellsurface localizing peptide sequences may be used. These sequencespreferably have glycosaminoglycan binding ability and, most preferably,bind heparin. Such peptide sequences may be derived from proteins havingheparin binding activity including, but not limited to, the following:protease nexin-1, protease nexin-2, antithrombin III, protein Cinhibitor, platelet factor 4, heparin cofactor II, ghilanten-relatedinhibitors, and bovine pancreatic trypsin inhibitor. Appropriateportions of these proteins (i.e. those with glycosaminoglycan bindingactivity) may be attached in the A or C position (or both).

In the case of TFPI, the appropriate portion may be the C-terminal tailSEQ ID NO: 7! or

    __________________________________________________________________________    Lys                                                                              Thr                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Lys                                                                              Gln                                                                              Arg                                                                              Val                                                                              Lys                                                                              Ile                                                                              Ala                                                                              Tyr                                                                              Glu                              Glu                                                                              Ile                                                                              Phe                                                                              Val                                                                              Lys                                                                              Asn                                                                              Met.                                                                              SEQ ID NO: 10!.                                         __________________________________________________________________________

In the case of TFPI-2, the appropriate portion may be the C-terminaltail SEQ ID NO: 8! or

    __________________________________________________________________________    Lys                                                                              Lys                                                                              Lys                                                                              Lys                                                                              Lys                                                                              Met                                                                              Pro                                                                              Lys                                                                              Leu                                                                              Arg                                                                              Phe                                                                              Ala                                                                              Ser                                                                              Arg                                                                              Ile                                                                              Arg                              Lys                                                                              Ile                                                                              Arg                                                                              Lys                                                                              Lys                                                                              Gln                                                                              Phe.                                                                              SEQ ID NO: 11!.                                         __________________________________________________________________________

In the case of antithrombin III, the appropriate portion may be

    __________________________________________________________________________    Ala                                                                              Lys                                                                              Leu                                                                              Asn                                                                              Cys                                                                              Arg                                                                              Leu                                                                              Tyr                                                                              Arg                                                                              Lys                                                                              Ala                                                                              Asn                                                                              Lys                                                                              Ser                                                                              Ser                                                                              Lys                              Leu.                                                                              SEQ ID NO: 12!.                                                           __________________________________________________________________________

The appropriate portion of antithrombin III may also be Thr Set Asp GinIle His Phe Phe Phe Ala Lys Leu Ash Cys Arg. SEQ ID NO: 13!

In the case of protein C inhibitor, the appropriate portion may be:

    __________________________________________________________________________    Ser                                                                              Glu                                                                              Lys                                                                              Thr                                                                              Leu                                                                              Arg                                                                              Lys                                                                              Trp                                                                              Leu                                                                              Lys                                                                              Met                                                                              Phe                                                                              Lys                                                                              Lys                                                                              Arg                                                                              Glu                              Leu                                                                              Glu                                                                              Glu                                                                              Tyr.                                                                              SEQ ID NO: 14!.                                                  __________________________________________________________________________

The appropriate portion of protein C inhibitor may be His Arg His HisPro Arg Glu Met Lys Lys Arg Val Glu Asp Leu. SEQ ID NO: 15!.

In the case of heparin cofactor II, the appropriate portion may be

    __________________________________________________________________________    Phe                                                                              Arg                                                                              Lys                                                                              Leu                                                                              Thr                                                                              His                                                                              Arg                                                                              Leu                                                                              Phe                                                                              Arg                                                                              Arg                                                                              Asn                                                                              Phe                                                                              Gly                                                                              Tyr                                                                              Thr                              Leu                                                                              Arg.                                                                              SEQ ID NO: 16!.                                                        __________________________________________________________________________

In the case of platelet factor 4, the appropriate portion may be Leu TyrLys Lys Ile Leu Lys Lys Leu Leu Glu Ala. SEQ ID NO: 17!.

In the case of ghilanten-related inhibitors, the appropriate portion maybe

    __________________________________________________________________________    Asn                                                                              Gly                                                                              Leu                                                                              Lys                                                                              Arg                                                                              Asp                                                                              Lys                                                                              Leu                                                                              Gly                                                                              Cys                                                                              Glu                                                                              Tyr                                                                              Cys                                                                              Glu                                                                              Cys                                                                              Arg                              Pro                                                                              Lys                                                                              Arg                                                                              Lys                                                                              Leu                                                                              Ile                                                                              Pro                                                                              Arg                                                                              Leu                                                                              Ser.                                                                              SEQ ID NO: 18!.                                __________________________________________________________________________

In a preferred embodiment, a factor VIIa/TF/X_(a) binding proteincontains the first Kunitz-type domain SEQ ID NO: 2!, including theamino-terminal sequence, of TFPI-2, the second Kunitz-type domain ofTFPI SEQ ID NO: 3!, the third Kunitz-type domain of TFPI SEQ ID NO: 5!and/or the TFPI C terminal tail sequence SEQ ID NO: 7!. One skilled inthe art will appreciate that this molecule is but one of numerousspecies that may be produced and that various portions of the peptidesequences linking the Kunitz-type domains from TFPI or from TFPI-2 maybe included in the molecule.

Also within the scope of the invention are factor VIIa/TF/X_(a) bindingproteins containing two or more of the same Kunitz-type domain from TFPIor from TFPI-2. For example, factor VIIa/TF/X_(a) binding proteinscomprising two or more iterations of the first Kunitz-type domain ofTFPI-2 SEQ ID NO: 2! may prepared. Such molecules may be particularlyuseful for increased inhibition of factor VIIa/TF complex. FactorVIIa/TF/X_(a) binding proteins containing two or more iterations of thesecond Kunitz-type domain of TFPI SEQ ID NO: 3! may also be prepared.Such molecules may be particularly useful for the increased inhibitionof factor X_(a) by the protein. A preferred factor VIIa/TF/Xa bindingprotein containing more than one iteration of the same Kunitz-typedomain is represented by the formula:

    A- X.sub.1 -B-X.sub.2 !.sub.c -C

wherein A and C are optional flanking peptides, the flanking peptidesindependently containing 1-100 amino acids;

wherein B is an optional spacer peptide, the spacer peptide containing1-25 amino acids;

wherein each X₁ is -D-K₁ -E-

where D,E are peptides of 1-25 amino acids,

where K₁ is Kunitz-type domain 1 from TFPI or TFPI-2 SEQ ID NO: 1 or SEQID NO: 2! or a mutein of the aforementioned Kunitz-type domains;

wherein each X₂ is -F-K₂ -G-

where F,G are peptides of 1-25 amino acids,

where K₂ is Kunitz-type domain 2 from TFPI or TFPI-2 SEQ ID NO: 3 or SEQID NO: 4! or a mutein of the aforementioned Kunitz-type domains;

wherein c is an integer from 1-10.

A, B, C, D, E, F, and G may also have the same sequences disclosed abovein reference to the generic structure. The factor VIIa/TF/Xa bindingprotein may be one in which K₁ and K₂ are SEQ ID NO: 2 and SEQ ID NO: 3.

One skilled in the art of DNA cloning and in possession of the DNAencoding TFPI and TFPI-2 is able to prepare suitable DNA molecules forproduction of such chimeric proteins using known cloning procedures(e.g. restriction enzyme digestion of TFPI and TFPI-2 encoding DNA,exonuclease digestion, ligation, and other appropriate proceduresoutlined in any of the following: Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL 2nd ed. (Cold Spring Harbor Laboratory Press, 1989);DNA CLONING, Vol. I and II, D. N. Glover ed. (IRL Press, 1985);OLIGONUCLEOTIDE SYNTHESIS, M. J. Gait ed. (IRL Press, 1984); NUCLEICACID HYBRIDIZATION, B. D. Hames & S. J. Higgins eds. (IRL Press, 1984);TRANSCRIPTION AND TRANSLATION, B. D. Hames & S. J. Higgins eds., (IRLPress, 1984); ANIMAL CELL CULTURE, R. I. Freshney ed. (IRL Press, 1986);IMMOBILIZED CELLS AND ENZYMES, K. Mosbach (IRL Press, 1986); B. Perbal,A PRACTICAL GUIDE TO MOLECULAR CLONING, Wiley (1984); the series,METHODS IN ENZYMOLOGY, Academic Press, Inc.; GENR TRANSFER VECTORS FORMAMMALIAN CELLS, J. H. Miller and M. P. Calos eds. (Cold Spring HarborLaboratory, 1987); METHODS IN ENZYMOLOGY, Vol. 154 and 155, Wu andGrossman, eds., and Wu, ed., respectively (Academic Press, 1987),IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY, R. J. Mayer and J.H. Walker, eds. (Academic Press London, Harcourt Brace U.S., 1987),PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed. (Springer-Verlag,N.Y. (1987), and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Vol. I-IV, D. M.Weir et al., (Blackwell Scientific Publications, 1986); Kitts et al.,Biotechniques 14:810-817 (1993 ); Munemitsu et al., Mol. Cell. Biol.10:5977-5982 (1990). Alternatively, the entire sequence or portions ofnucleic acid sequences encoding proteins described above may be preparedby synthetic methods (e.g. using DNA synthesis machines). Finally, apreferred method of preparing nucleic acid molecules encoding thedescribed chimeric proteins is by use of PCR techniques as described inInnis et al, supra.

The proteins described above may be prepared using any suitableexpression system including, without limitation, the followingexpression systems: mammalian tissue culture, insect cell culture,bacterial cell culture and yeast cell culture. Mammalian expressionsystems are known in the art. Sambrook et al. (1989) "Expression ofCloned Genes in Mammalian Cells." In Molecular Cloning: A LaboratoryManual, 2nd ed. Mammalian cell lines available as hosts for expressionare known in the art and include many immortalized cell lines availablefrom the American Type Culture Collection (ATCC). TFPI has been isolatedfrom human plasma and from human tissue culture cells including HepG2,Chang liver and SK hepatoma cells. Recombinant TFPI has been expressedin mouse C127 cells, baby hamster kidney cells, Chinese hamster ovarycells and human SK hepatoma cells.

The proteins of the invention may also be produced in insect cells usinga vector containing baculovirus sequences. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit).These techniques are generally known to those skilled in the an andfully described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987) hereinafter "Summers and Smith").Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 base pairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith supra; Ju et al.(1987), Smith et al, Mol. Cell. Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., Bioessays 4:91 (1989). The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5' and 3' by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti, Bombyxmori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusiani (PCT Pub. No. WO 89/046699; Carbonell et al., J. Virol. 56:153(1985); Wright Nature 321:718 (1986); Smith et al., Mol. Cell. Biol.3:2156 (1983); and see generally, Fraser, et al. Cell. Dev. Biol. 25:225(1989). Cells and cell culture media are commercially available for bothdirect and fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, e.g., Summers and Smith supra.The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. A presently preferred media is described inEPO 380 495.

Numerous bacterial expression techniques are known in the art. Sambrooket al. (1989) "Expression of cloned genes in Escherichia coli." InMolecular Cloning: A Laboratory Manual. Expression and transformationvectors, either extra-chromosomal replicons or integrating vectors, havebeen developed for transformation into many bacteria. For example,expression vectors have been developed for, inter alia, the followingbacteria: Bacillus subtilis (Palva et al. (1982) Proc. Natl. Acad. Sci.USA 79:5582; EPO 036 259 and 063 953; PCT WO 84/04541), E. coli(Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EPO Publ. Nos. 036776, 136 829 and 136 907), Streptococcus cremoris (Powell et al. (1988)Appl. Environ. Microbiol. 54:655); Streptococcus lividanis (Powell etal. (1988) Appl. Environ. Microbiol. 54:655), Streptomyces lividanis(U.S. Pat. No. 4,745,056).

The DNA encoding the protein of the present invention may be joined to asignal peptide for export or secretion of the mature protein to theperiplasmic space of bacteria, using techniques that are conventional inthe art. Moreover, transcription and translation can further beoptimized in a bacterial expression system by varying the spacingbetween the DNA to be expressed and the sequences encoding the promoterand ribosome binding site.

Yeast expression systems are also known in the art. Fusion proteinsprovide one means for expression of the proteins of the invention inyeast systems. Usually, a DNA sequence encoding the N-terminal portionof an endogenous yeast protein, or other stable protein, is fused to the5' end of a heterologous coding sequence. Upon expression, thisconstruct will provide a fusion of the two amino acid sequences. The DNAsequence at the junction of the two amino acid sequences may or may notencode a cleavable site. See e.g., EPO 196 056. A preferred fusionprotein is a ubiquitin fusion protein. Such a fusion protein is madewith the ubiquitin region that preferably retains a site for aprocessing enzyme, which allows a ubiquitin-specific processing proteaseto cleave the ubiquitin from the foreign protein. Through this method,therefore, foreign protein with an authentic amino terminus can beisolated from within the yeast cell. Production of ubiquitin fusionproteins is described in co-pending U.S. patent application Ser. Nos.07/806,813 and 07/957,627. This method is reviewed in Barr et al, inRECOMBINANT SYSTEMS IN PROTEIN EXPRESSION (Elsevier Science PublishersB.V., 1991), pp. 37-46.

Alternatively, foreign proteins can be secreted from the cell into thegrowth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (EP 012 873;JPO 62,096,086), the α-factor gene (U.S. Pat. Nos. 4,588,684 and4,870,008; EP 116,201) and truncated versions of the α-factor gene asdescribed in EP 324 274 and co-pending U.S. patent application Ser. No.07/864,206. Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast (EP060 057). Preferably, the α-factor gene is used in nucleic acidconstructs designed for secretion of the proteins of the invention.

Another useful class of secretion leaders are those that employ afragment of the yeast α-factor gene, which contains both a "pre" signalsequence, and a "pro" region. The types of α-factor fragments that canbe employed include the full-length pre-pro a factor leader (about 83amino acid residues) as well as truncated α-factor leaders (usuallyabout 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and4,870,008; EP 324 274). Additional leaders employing an α-factor leaderfragment that provides for secretion include hybrid α-factor leadersmade with a presequence of a first yeast, but a pro-region from a secondyeast α-factor. (See e.g., PCT WO 89/02463.)

Expression vectors encoding the proteins of the invention are oftenmaintained in a replicon, such as an extrachromosomal element (e.g.,plasmid) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprocaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein et al, Gene8:17-24 (1979)), pCl/1 (Brake et al, Proc. Natl. Acad. Sci USA81:4642-4646 (1984)), and YRp17 (Stinchcomb et al, J. Mol. Biol. 158:157(1982)). In addition, a replicon may be either a high or low copy numberplasmid. A high copy number plasmid will generally have a copy numberranging from about 5 to about 200, and usually about 10 to about 150. Ahost containing a high copy number plasmid will preferably have at leastabout 10, and more preferably at least about 20. Either a high or lowcopy number vector may be selected, depending upon the effect of thevector and the foreign protein on the host. See e.g., Brake et al,supra. For production of the proteins of the invention in a yeast cellwherein the protein is retained within the yeast cell, a plasmid such aspAB24 may be used. Sabin et al, (1989) Bio/Technology 7:705-709. pAB24contains a GAP/ADH hybrid promoter, containing portions of an ADHpromoter capable of directing high levels of expression of the sequencesunder its control but which also contains GAP regulatory sequences,allowing expression of the same sequence a desired point in the growthof a culture.

Alternatively, the expression constructs can be integrated into the hostgenome with an integrating vector. Integrating vectors usually containat least one sequence homologous to a host chromosome that allows thevector to integrate, and preferably contain two homologous sequencesflanking the expression construct. Integrations appear to result fromrecombinations between homologous DNA in the vector and the hostchromosome Orr-Weaver et al, Meth. Enzymol. 101:228-245 (1983). Anintegrating vector may be directed to a specific locus in yeast byselecting the appropriate homologous sequence for inclusion in thevector. See Orr-Weaver et al, supra. One or more expression constructmay integrate, possibly affecting levels of recombinant proteinproduced. Rine et al, Proc. Natl. Acad. Sci. USA 80:6750 (1983). Thechromosomal sequences included in the vector can occur either as asingle segment in the vector, which results in the integration of theentire vector, or two segments homologous to adjacent segments in thechromosome and flanking the expression construct in the vector, whichcan result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP₁, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP 1 allows yeast to grow in the presence of copper ions(Butt et al, Microbiol. Rev. 51:351 (1987)).

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans (Kurtz, et al, Mol. Cell.Biol. 6:142 (1986)), Candida maltosa (Kunze, et al, J. Basic Microbiol.25:141 (1985)), Hansenula polymorpha (Gleeson, et al, J. Gen. Microbiol.132:3459 (1986); Roggenkamp et al, Mol. Gen. Genet. 202:302 (1986)),Kluyveromyces fragilis (Das, et al, J. Bacteriol. 158:1165 (1984)),Kluyveromyces lactis (De Louvencourt et al, J. Bacteriol. 154:737(1983); Van den Berg et al, Bio/Technology 8:135 (1990)), Pichiaguillerimondii (Kunze et al, J. Basic Microbiol. 25:141 (1985)), Pichiapastoris (Cregg, et al, Mol. Cell. Bio. 5:3376 (1985); U.S. Pat. Nos.4,837,148 and 4,929,555), Saccharomyces cerevisiae (Hinnen et al, Proc.Natl. Acad. Sci. USA 75:1929 (1978); Ito et al, J. Bacteriol. 153:163(1983)), Schizosaccharomyces pombe (Beach and Nurse, Nature 300:706(1981)), and Yarrowia lipolytica (Davidow, et al, Curr. Genet. 10:380471(1985) and Gaillardin et al, Curt. Genet. 10:49 (1985)).

Transformation procedures that may be used herein to transform yeastcells include electroporation, as described in "Guide to Yeast Geneticsand Molecular Biology," Vol 194 METHODS IN ENZYMOLOGY, C. Guthrie and G.R. Fink, (Academic Press 1991). Other procedures include thetransformation of spheroplasts or the transformation of alkalication-treated intact cells. Such procedures are described in, forexample, Kurtz et al, Mol. Cell. Biol. 6:142 (1986); Kunze et al, J.Basic Microbiol. 25:141 (1985), for Candida; Gleeson et al, J. Gen.Microbiol. 132:3459 (1986); Roggenkamp et al., Mol. Gen. Genet. 202:302,for Hansenula (1986); Das et al, J. Bacteriol. 158:1165 (1984); DeLouvencourt et al, J. Bacteriol. 154:1165 (1983); Van den Berg et al,Bio/Technology 8:135 (1990) for Kluyveromyces; Cregg et al, Mol. Cell.Biol. 5:3376 (1985); Kunze et al, J. Basic Microbiol. 25:141 (1985);U.S. Pat. Nos. 4,837,148 and 4,929,555, for Pichia; Hinnen et al, Proc.Natl. Acad. Sci. USA 75:1929 (1978); lto et al, J. Bacteriol. 153:163(1983), for Saccharomyces; Beach and Nurse, Nature 300:706 (1981 ), forShizosaccharomyces; Davidow et al, Curr. Genet. 10:39 (1985); Gaillardinet al, Curr. Genet. 10:49 (1985), for Yarrowia.

Yeast cell culture, especially Saccharomyces cerevisiae, are preferredfor production of the proteins of the invention. In a preferredembodiment, the chimeric protein having the primary amino acid sequence:

    __________________________________________________________________________    Asp                                                                              Ser                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Glu                                                                              Glu                                                                              His                                                                              Thr                                                                              Ile                                                                              Ile                                                                              Thr                                                                              Asp                                                                              Thr                                                                              Glu                                                                              Leu                              Pro                                                                              Pro                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Met                                                                              His                                                                              Ser                                                                              Phe                                                                              Cys                                                                              Ala                                                                              Phe                                                                              Lys                                                                              Ala                                                                              Asp                                                                              Asp                              Gly                                                                              Pro                                                                              Cys                                                                              Arg                                                                              Ala                                                                              Ile                                                                              Met                                                                              Lys                                                                              Arg                                                                              Phe                                                                              Phe                                                                              Phe                                                                              Asn                                                                              Ile                                                                              Phe                                                                              Thr                              Arg                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Glu                                                                              Phe                                                                              Ile                                                                              Tyr                                                                              Gly                                                                              Gly                                                                              Cys                                                                              Glu                                                                              Gly                                                                              Asn                                                                              Gln                                                                              Asn                              Arg                                                                              Phe                                                                              Glu                                                                              Ser                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                                                                              Lys                                                                              Met                                                                              Cys                                                                              Thr                                                                              Arg                                                                              Asp                                                                              Asn                              Ala                                                                              Asn                                                                              Arg                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Thr                                                                              Thr                                                                              Leu                                                                              Gln                                                                              Gln                                                                              Glu                                                                              Lys                                                                              Pro                                                                              Asp                                                                              Phe                              Cys                                                                              Phe                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Pro                                                                              Gly                                                                              Ile                                                                              Cys                                                                              Arg                                                                              Gly                                                                              Tyr                                                                              Ile                                                                              Thr                                                                              Arg                              Tyr                                                                              Phe                                                                              Tyr                                                                              Asn                                                                              Gln                                                                              Gln                                                                              Thr                                                                              Lys                                                                              Gln                                                                              Cys                                                                              Glu                                                                              Arg                                                                              Phe                                                                              Lys                                                                              Tyr                                                                              Gly                              Gly                                                                              Cys                                                                              Leu                                                                              Gly                                                                              Asn                                                                              Met                                                                              Asn                                                                              Asn                                                                              Phe                                                                              Glu                                                                              Thr                                                                              Leu                                                                              Glu                                                                              Glu                                                                              Cys                                                                              Lys                              Asn                                                                              Ile                                                                              Cys                                                                              Glu                                                                              Asp                                                                              Gly                                                                              Pro                                                                              Asn                                                                              Gly                                                                              Phe                                                                              Gln                                                                              Val                                                                              Asp                                                                              Asn                                                                              Tyr                                                                              Gly                              Thr                                                                            SEQ ID NO: 19!                                                               __________________________________________________________________________

is produced in a yeast cell as a fusion protein with α-factor.

Alternatively, the flanking peptides may contain sequences derived fromother protease inhibitors, especially protein inhibitors which act atthe cell surface. The protein inhibitor protease nexin 1 (hereinafter,nexin) is but one example. Nexin is known to bind heparin therebyhelping to localize nexin on the cell surface. Further, nexin binds toand inhibits factor Xa.

The factor VIIa/TF/X_(a) binding proteins of the invention may beassayed for activity by a prothrombin time clotting assay or a factor Xaamidolytic assay (Wun et al, J. Biol. Chem 265:16096 (1990)) as setforth in the Examples below.

Formulation and Administration

Factor VIIa/TF/X_(a) binding proteins of the invention may beadministered at a concentration that is therapeutically effective totreat and prevent septic shock. To accomplish this goal, the factorVIIa/TF/X_(a) binding proteins of the invention are preferablyadministered intravenously. Methods to accomplish this administrationare known to those of ordinary skill in the art.

Before administration to patients, formulants may be added to the factorVIIa/TF/Xa binding proteins of the invention. A liquid formulation maybe used. For example, these formulants may include oils, polymers,vitamins, carbohydrates, amino acids, salts, buffers, albumin,surfactants, or bulking agents. Carbohydrates which may be used in theformulation include sugar or sugar alcohols such as mono, di, orpolysaccharides, or water soluble glucans. The saccharides or glucanscan include fructose, dextrose, lactose, glucose, mannose, sorbose,xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and betacyclodextrin, soluble starch, hydroxethyl starch andcarboxymethylcellulose, or mixtures thereof. Sucrose is most preferred.Sugar alcohol is defined as a C₄ to C₈ hydrocarbon having an --OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. Mannitol is most preferred. These sugars orsugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to amount used as long as the sugaror sugar alcohol is soluble in the aqueous preparation. Preferably, thesugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %,more preferable between 2.0 and 6.0 w/v %. Preferably amino acidsinclude levorotary (L) forms of carnitine, arginine, and betaine;however, other amino acids may be added. Preferred polymers includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000. It is also preferred to use a buffer inthe composition to minimize pH changes in the solution beforelyophilization or after reconstitution. Most any physiological buffermay be used, but citrate, phosphate, succinate, and glutamate buffers ormixtures thereof are preferred. Most preferred is a citrate buffer.Further, the use of sulfates should be avoided in preparation of theformulation. Preferably, the concentration is from 0.01 to 0.3 molar.Surfactants that can be added to the formulation are shown in EP Nos.270,799 and 268,110.

Additionally, the factor VIIa/TF/Xa binding proteins of the inventioncan be chemically modified by covalent conjugation to a polymer toincrease its circulating half-life, for example. Preferred polymers, andmethods to attach them to peptides, are shown in U.S. Pat. Nos.4,766,106, 4,179,337, 4,495,285, and 4,609,546 which are all herebyincorporated by reference in their entireties. Preferred polymers arepolyoxyethylated polyols and polyethyleneglycol (PEG). PEG is soluble inwater at room temperature and has the general formula: R(O--CH₂--CH₂)_(n) O--R where R can be hydrogen, or a protective group such asan alkyl or alkanol group. Preferably, the protective group has between1 and 8 carbons, more preferably it is methyl. The symbol n is apositive integer, preferably between 1 and 1,000, more preferablybetween 2 and 500. The PEG has a preferred average molecular weightbetween 1000 and 40,000, more preferably between 2000 and 20,000, mostpreferably between 3,000 and 12,000. Preferably, PEG has at least onehydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/factor VIIa/TF/Xa of the present invention.

Water soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), etc. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has apreferred molecular weight in the same range as PEG. The structure forPOG is shown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070, and adiscussion of POG/protein conjugates is found in U.S. Pat. No.4,766,106, both of which are hereby incorporated by reference in theirentireties.

After the liquid pharmaceutical composition is prepared, it ispreferably lyophilized to prevent degradation and to preserve sterility.Methods for lyophilizing liquid compositions are known to those ofordinary skill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is preferablyadministered to subjects using those methods that are known to thoseskilled in the art.

Administration to Affected Individuals

Factor VIIaITF/Xa binding proteins of the invention are useful to treatmammals with sepsis or septic shock. Generally, conditions arecharacterized by high fever (>38.5° C.) or hypothermia (>35.5° C.), lowblood pressure, tachypnea (>20 breaths/minute), tachycardia (>100beats/minute), leukocytosis (>15,000 cells/mm³) and thrombocytopenia(<100,000 platelets/mm3). The factor VIIa/TF/Xa binding proteins of theinvention are preferably administered as soon as the subject issuspected of being septic; presenting a >20% drop in fibrinogen orappearance of fibrin split products, a rise in the subject's temperatureand the diagnosis of leukopenia and hypotension associated with septicshock. As stated above, intravenous administration is preferred.Generally, factor VIIa/TF/Xa binding proteins of the invention are givenat a dose between 1 μg/kg and 20 mg/kg, more preferably between 20 μg/kgand 10 mg/kg, most preferably between 1 and 7 mg/kg. Preferably, it isgiven as a bolus dose, to increase circulating levels by 10-20 fold andfor 4-6 hours after the bolus dose. Continuous infusion may also be usedafter the bolus dose. If so, the factor VIIa/TF/Xa binding proteins ofthe invention may be infused at a dose between 5 and 20 μg/kg/minute,more preferably between 7 and 15 μg/kg/minute.

The factor VIIa/TF/Xa binding proteins of the invention may be given incombination with other agents which would be effective to treat septicshock. For example, the following may be administered in combinationwith the factor VIIa/TF/Xa binding proteins of the invention:antibiotics that can treat the underlying bacterial infection;monoclonal antibodies that are directed against bacterial cell wallcomponents; monoclonal antibodies and soluble receptors that can complexwith cytokines that are involved in the sepsis pathway, including, butnot limited to tumor necrosis factor (TNF), Interleukin-1, γ-interferonand interleukin-8; and generally any agent or protein that can interactwith cytokines or complement proteins in the sepsis pathway to reducetheir effects and to attenuate sepsis or septic shock.

Antibiotics useful in the present invention include those in the generalcategory of: beta-lactam rings (penicillin), amino sugars in glycosidiclinkage (amino glycosides), macrocyclic lactone rings (macrolides),polycyclic derivatives of napthacenecarboxamide (tetracyclines),nitrobenzene derivatives of dichloroacetic acid, peptides (bacitracin,gramicidin, and polymyxin), large rings with a conjugated double bondsystem (polyenes), sulfa drugs derived from sulfanilarnide(sulfonamides), 5-nitro-2-furanyl groups (nitrofurans), quinolonecarboxylic acids (nalidixic acid), and many others. Other antibioticsand more versions of the above specific antibiotics may be found inEncyclopedia of Chemical Technology, 3rd Edition, Kirk-Othymer (ed.),Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78, Zinsser,MicroBiology, 17th Edition W. Joklik et al (Eds.) pages 235-277 (1980),or Dorland's Illustrated Medical Dictionary, 27th Edition, W. B.Saunders Company (1988).

Other agents which may be combined with the factor VIIa/TF/Xa bindingproteins of the invention include monoclonal antibodies directed tocytokines involved in the sepsis pathway, such as those monoclonalantibodies directed to IL-6 or M-CSF, such as shown in PCT US90/07411;monoclonal antibodies directed to TNF, such as shown in U.S. Pat. No.4,603,106; inhibitors of proteins that cleave the mature TNF prohormonefrom the cell in which it was produced, such as shown in PCT US90/03266and PCT US93/06120; antagonists of IL-1, such as shown in PCTUS91/02460; inhibitors of IL-6 cytokine action such as activin, such asshown in PCT US90/00321; and receptor based inhibitors of variouscytokine such as IL-1. Antibodies to or small molecule inhibitors ofcomplement protein may also be employed.

Generally, the factor VIIa/TF/Xa binding proteins of the invention maybe useful for those diseases that occur due to the up-regulation oftissue factor brought on by injury, trauma, endotoxin, TNF, cancer, IL-1or other agents or conditions.

EXAMPLES

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

Example 1

The shuttle vector pBS24 is described in Barr et al, EXPRESSION SYSTEMS& PROCESSES FOR rDNA PRODUCTS (American Chemical Society, 1991 ), pp51-64). pBS24Ub is a derivative of pBS24.1, and contains an expressioncassette flanked by unique Barn HI and Sal I restriction sites, theglucose regulatable ADH2/GAP promoter and a synthetic ubiquitin (Ub)gene. For construction of Ub fusions, a unique SstII site is generatedin the 3' end of the Ub gene. The presence of the SstII site allowsin-frame insertion of nucleotide sequences for expression as ubiquitinfusion peptides. Insertion can be accomplished by use of synthetic DNAadapters or PCR methodologies. Methods for using synthetic DNA adapters(linkers) are known in the art. All enzymatic modifications of DNA andprotein are done according to the instructions provided by themanufacturer of the enzyme. PCR protocols are described in PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, (eds.) Innis, Gelfand,Sninsky and White (Academic Press, 1990). In either case, the5'-junction sequence will be: ##STR1## and the 3' cloning site (Sal I)should be as close as possible to the 3' end of the termination codon.

PCR was used to construct the ubiquitin/TFPI gene fusion in the 15.4 kbplasmid pLACI 4.1 shown in FIG. 1. TFPI encoding nucleic acid wasamplified using standard PCR procedures with the primers SEQ ID NO: 21and SEQ ID NO: 22. SEQ ID NO: 21 hybridizes to the 10 nucleotides at the5' end of nucleic acid mature encoding TFPI and also contains ubiquitinsequence with the Sst II restriction site. SEQ ID NO: 22 hybridizes tothe 15 nucleotides at the 3' end of nucleic acid encoding mature TFPIand also trailing sequence with a Sal I restriction site. The sequencesof these primers are as follows:

SEQ ID NO: 21 GCTCCGCGGTGGCGATTCTGAGGAGGAGATGAAGAAC

SEQ ID NO: 22 TCTGTCGACTCACATATTTTTAACAAAAATTTCTTCAT

After amplification, the PCR product was digested with Sal I and Sst IIusing conditions specified by the manufacturer of the enzymes. Thedigested PCR product was then cloned into pBS24Ub, as described above,to produce pLACI4.1. S. cerevisiae strain AB 122 transformed withpLACI4.1 has was deposited with the ATCC on Jul. 19, 1994 and has beengiven Accession Number 74291.

pLACI 4.1 was used to transform three strains of Saccharomycescerevisiae: VH6 (MAT α, cir°, leu-2-112,-3, ura3, FoA, pep4::His3),AB122 (MAT α, cir°, leu2, ura3-52, prb1-1112, pep4-3, prc1:407) andJSC310 (as AB122,+ADR1 overexpression). Transformants of VH6 producedTFPI at levels of approximately 5% of total protein, transformants of AB122 produced TFPI at levels of approximately 10% of total protein andtransformants of JSC310 produced TFPI at levels of approximately 15% oftotal protein. TFPI expressed according to this method was shown to havebiological activity, that is TFPI showed both factor VIIa/TF and factorXa inhibition.

Example 2

TFPI was expressed as a full length fusion protein with prepro α factorleader. The α-factor/TFPI fusion protein was constructed using pAB125 asan intermediate plasmid. (Chang et al, J. Immunol. 149:548-555 (1992)).pAB 125 contains an expression cassette flanked by unique Bam HI and SalI restriction sites, the glucose regulatable ADH2/GAP promoter and theα-factor prepro leader sequence and processing site. For construction ofα-factor fusions, a unique Xba I site is generated in the 3' end of theα factor leader gene sequence. The presence of the Xba I site allowsin-frame insertion of nucleotide sequences for expression as a factorfusion peptides. Insertion can be accomplished by use of synthetic DNAadapters or PCR methodologies. In either case, the 5' junction sequencewill be ##STR2## and the 3' cloning site (Sal I) should be as close aspossible to the 3' end of the termination codon.

PCR was used to construct the α-factor/TFPI gene fusion in the 15.4 kbplasmid pLACI2.1. TFPI encoding nucleic acid was amplified usingstandard PCR procedures with the primers SEQ ID NO: 24 and SEQ ID NO:22. SEQ ID NO: 24 hybridizes to the 22 nucleotides at the 5' end ofnucleic acid encoding mature TFPI and also contains α-factor sequencewith the Xha I restriction site as shown above. 22 hybridizes to the 29nucleotides at the 3' end of nucleic acid encoding mature TFPI andtrailing sequence with a Sal I restriction site. The sequence of SEQ IDNO: 24 is as follows:

SEQ ID NO: 24 ATCTCTAGATAAAAGAGATTCTGAGGAAGATGAAGAAC

After amplification, the PCR product was digested with Sal I and Xba Iusing conditions specified by the manufacturer of the enzymes. Thedigested PCR product was then cloned into pAB125 which had beenpreviously digested with Sal I and Xba I. The Bam HI to Sal I fragmentof pAB125 containing the α-factor/TFPI fusion protein was isolated andsubcloned into pBS24.1 to produce pLACI2.1

pLACI2.1 was used to transform the VH6 and AB122 strains of S.cerevisiae.

Example 3

Truncated TFPI, containing amino acids 1-116 of mature TFPI, wasexpressed as an α-factor fusion protein for secretion. Preparation ofTFPI-encoding sequence was accomplished essentially as in Example 2using SEQ ID NO: 24 and a second primer, SEQ ID NO: 25, which hybridizeswithin the TFPI coding sequence. The sequence of SEQ ID NO: 25 is asfollows:

SEQ ID NO: 25 TCTGTCGACTCAGGTTCCATAATTATCCACCT

Alternatively, truncated TFPI can be expressed as a ubiquitin fusionprotein and recovered from within the yeast cell. In order to preparethe appropriate coding sequence, the LACI4 and LACI2 primers are used toprepare the coding region for subcloning into pBS24-Ub as describedabove.

Example 4

Yeast shake flask cultures were harvested by centrifugation and thesupernatant fluids containing α-factor-TFPI were collected and filteredthrough 0.81μ membranes. The cells from TFPI cultures were pelleted bycentrifugation. Cell lysates from α-factor-TFPI and ubiquitin-TFPI wereprepared by disrupting cell pellets in 50 mM Tris pH 7.5, 2 mM EDTA, 50mM NaCl, 1 mM PMSF by vortexing with glass beads. In some cases, thecell lysates were further fractionated into soluble and insolublefractions by centrifugation at 12000 g for 10 minutes at 4° C.

Expression of TFPI was detected by SDS/PAGE followed by Coomassiestaining or by Western blotting with a rabbit antiserum raised againstthe first 15 amino acids of TFPI (NTP sera). Analysis of the soluble andinsoluble fractions of cell lysates is shown in FIGS. 2A and 2B.Coomassie-stained gels (top panel) reveal the presence of unique bandsmigrating at ˜21 kD in the soluble fraction of α-factor-truncated TFPI(aa 1-161) (Lane 2, FIG. 2A and FIG. 2B) and insoluble fractions ofubiquitin-truncated TFPI (aa 1-161) (Lane 5, FIG. 2A and FIG. 2B) whichwere verified to represent truncated TFPI by Western blotting (Lanes 2and 5, FIG. 2B). Full-length TFPI migrating at 35 kD was detectable inthe insoluble fraction of ubiquitin-TFPI lysates (Lane 6, FIG. 2A andFIG. 2B) by Coomassie staining and Western blotting and was identifiedin the soluble fraction of α-factor TFPI lysates by Western blotting.(Lane 3, FIG. 2B). Lanes 1 and 4 of FIGS. 2A and 2B contained negativecontol proteins isolated as above from non-transformed yeast cellculture.

Expression of secreted TFPI was detected by analysis of culturesupernates. Biochemical analysis of protein expression included SDS/PAGEfollowed by Coomassie staining or Western blotting with NTP sera. Sincetruncated TFPI may be N-glycosylated at one site and full-length TFPIhas the potential for modification by N-glycosylation at three sites,the supernatant samples were concentrated five-fold, dialyzed againstTris buffer and deglycosylated with N-glycanase according to themanufacturer's recommendations (Genzyme). As shown in FIG. 3, truncatedTFPI was easily detectable by Coomassie staining and by Western blottingas major bands migrating at ˜21 and 25 kD (Lane 2, FIG. 3A and FIG. 3B).The 25 kD band was verified as representing glycosylated truncated TFPIby conversion of the doublet to the single 21 kD band followingN-glycanase digestion (Lane 5, FIG. 3A and FIG. 3B). While secretedfull-length TFPI was not as readily identified prior to N-glycanasetreatment (Lane 3, FIG. 3A and FIG. 3B), the 35 kD TFPI band could beobserved following deglycosylation (Lane 6, FIG. 3A and FIG. 3B). Lanes1 and 4 of FIGS. 3A and 3B contained negative contol proteins isolatedas above from non-transformed yeast cell culture.

Biological assays were performed using supernatants and solublefractions of cell lysates of TFPI cultures. A prothrombin time clottingassay (Wun et al, J. Biol. Chem. 265:16096 (1990)) indicated weak TFPIactivity in unconcentrated supernatants from cultures secretingtruncated TFPI, while the soluble fraction of intracellularly expressedtruncated and full-length TFPI exhibited TFPI activity as compared tothe intracellular control. A more sensitive assay of TFPI activity, theinhibition of factor Xa activity (Wun et al, ibid) was performed onsupernates from cultures producing truncated and full-length TFPI. Asshown in FIG. 4, significant Xa inhibitory activity was evident insupernates from cultures secreting truncated TFPI (L161) and full lengthTFPI (L-FL) as compared to negative control (V2) yeast culture.

Example 5

Potential sites for N-linked glycosylation within TFPI are removed usingoverlapping PCR as described in Innis et al, supra. As in the previousexamples, TFPI-encoding sequences are prepared by PCR, cloned in pAB125and further subcloned in pBS24.1 for expression.

For replacement of the asparagine at position 116 with glutamine, thefollowing overlapping primers are used:

SEQ ID NO: 26 AGGTATTTTTATAACAATCAGACAAAACAGTGT

SEQ ID NO: 27 GAAACGTTCACACTGTTTTGTCTGATTGTTATA

For replacement of the asparagine at position 167 with glutamine, thefollowing overlapping primers are used:

SEQ ID NO: 28 CCAGCTCAATGCTGTGAATAACTCCCTGACTCCG

SEQ ID NO: 29 CTTGGTTGATTGCGGAGTCAGGGAGTTATTCACAGC

For replacement of the asparagine at position 227 with glutamine, thefollowing overlapping primers are used:

SEQ ID NO: 30 GGGGGAAATGAAAACAATTTTACTTCCAAACAA

SEQ ID NO: 31 CCTCAGACATTCTTGTTTGGAAGTAAAATTGTTTTC

In each case, the LACI1 and LACI3 primers are used to provide thenecessary Xba I and Sal I sites for cloning. To introduce more than onemutation, thereby eliminating another potential glycosylation site,sequential rounds of overlapping PCR may be used.

Example 6

In order to produce a mutein of TFPI containing a Lys to Argsubstitution in the P1 reactive site of Kunitz-type domain 1, a BglII-Sal I fragment from pBS24Ub/TFPI1 was subcloned into pSP₇₂(commercially available from Promega). A Ava III-Bsp HI fragment wasthen prepared using the following primers:

SEQ ID NO: 32 CCGATGCATTCATTTTGTGCATTC

SEQ ID NO: 33 CCTCATGATTGCCCGACATGGGCC

The second primer contains a single mismatch from the native sequenceresulting in the desired Lys to Arg mutation in the resulting fragment.This mutated sequence is then used to replace the Ava III-Bsp HIfragment in the TFPI-encoding sequence previously cloned into pSP72. Theresulting plasmid is then digested with Sst II and Sal I and cloned intopBS24-Ub for expression.

Example 7

Constructs encoding chimeric TFPI proteins in which the Kunitz-typedomain 1 of TFPI is replaced with the Kunitz-type domain 1 of TFPI-2were prepared using overlapping PCR. A Sst II-Bsm I restrictionfragment, containing Kunitz-type domain 1 of TFPI-2 and part ofKunitz-type domain 2 of TFPI, was generated by overlapping PCR. Theprimers used were:

SEQ ID NO: 34 GGTCCGCGGTGGTGATGCTGCTCAGGAGC

SEQ ID NO: 35 GCAATGTTGTTTTTTCTATCCTCCAGCAAGCAT

These two primers were used to prepare a 93 bp fragment using the TFPI-2coding sequence as a template. The TFPI-2 coding sequence was clonedusing PCR. Primers for cloning were derived from the sequence publishedin Sprecher et al, Proc. Nat. Acad. Sci. USA 91:3353-3357 (1994).

A 54 bp fragment was then prepared using TFPI coding sequence as atemplate and the following primers:

SEQ ID NO: 36 GGATAGAAAAAAACAACATTGCAACAAGAAAAGC

SEQ ID NO: 37 GGTTCTTGCATTCTTCCAGTGTCTCAAAATTG

Overlapping PCR was then performed to join these two fragments using theprimers TFOLA and TFOLD. The 126 bp product was then digested with SstII and Bsm I and exchanged for the equivalent Sst I-Bsm I fragment inpSP72/TFP1. This construct was subsequently digested with Sst II and SalI and the resulting fragment cloned in pBS24-Ub for expression.

Example 8

Yeast strains are produced containing och1, mnn1 and alg3 mutationsresulting in reduction of carbohydrate attached to secreted TFPImolecules of the invention. The genes are cloned using standard PCRtechniques based on sequences available to one skilled in the art.

Deletions of 300 bp or more in the OCH1, MNN1 and ALG3 genes areintroduced into the coding region of each of these genes. The genes withsequences deleted are then cloned into URA3-based integrating vector.The wild type genes for each of the three are sequentially replacedusing the pop-in/pop-out replacement vector as described in Scherer andDavis, Proc. Nat. Acad. Sci. USA 76:4951 (1979).

The foregoing discussion and examples only illustrate the presentinvention, persons of ordinary skill in the art will appreciate that theinvention can be implemented in other ways, and the invention is definedsolely by reference to the claims. Further, all references, patents andpatent applications cited in the foregoing specification areincorporated herein by reference.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 37                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysAlaPheLysAlaAspAspGlyProCysLysAlaIleMetLysArg                              151015                                                                        PhePhePheAsnIlePheThrArgGlnCysGluGluPheIleTyrGly                              202530                                                                        GlyCysGluGlyAsnGlnAsnArgPheGluSerLeuGluGluCysLys                              354045                                                                        LysMetCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysLeuLeuProLeuAspTyrGlyProCysArgAlaLeuLeuLeuArg                              151015                                                                        TyrTyrTyrAspArgTyrThrGlnSerCysArgGlnPheLeuTyrGly                              202530                                                                        GlyCysGluGlyAsnAlaAsnAsnPheTyrThrTrpGluAlaCysAsp                              354045                                                                        AspAlaCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg                              151015                                                                        TyrPheTyrAsnAsnGlnThrLysGlnCysGluArgPheLysTyrGly                              202530                                                                        GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys                              354045                                                                        AsnIleCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CysArgLeuGlnValSerValAspAspGlnCysGluGlySerThrGlu                              151015                                                                        LysTyrPhePheAsnLeuSerSerMetThrCysGluLysPhePheSer                              202530                                                                        GlyGlyCysHisArgAsnArgIleGluAsnArgPheProAspGluAla                              354045                                                                        ThrCysMetGlyPheCys                                                            50                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CysLeuThrProAlaAspArgGlyLeuCysArgAlaAsnGluAsnArg                              151015                                                                        PheTyrTyrAsnSerValIleGlyLysCysArgProPheLysTyrSer                              202530                                                                        GlyCysGlyGlyAsnGluAsnAsnPheThrSerLysGlnGluCysLeu                              354045                                                                        ArgAlaCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysTyrSerProLysAspGluGlyLeuCysSerAlaAsnValThrArg                              151015                                                                        TyrTyrPheAsnProArgTyrArgThrCysAspAlaPheThrTyrThr                              202530                                                                        GlyCysGlyGlyAsnAspAsnAsnPheValSerArgGluAspCysLys                              354045                                                                        ArgAlaCys                                                                     50                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LysLysGlyPheIleGlnArgIleSerLysGlyGlyLeuIleLysThr                              151015                                                                        LysArgLysArgLysLysGlnArgValLysIleAlaTyrGluGluIle                              202530                                                                        PheValLysAsnMet                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaLysAlaLeuLysLysLysLysLysMetProLysLeuArgPheAla                              151015                                                                        SerArgIleArgLysIleArgLysLysGlnPhe                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 276 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AspSerGluGluAspGluGluHisThrIleIleThrAspThrGluLeu                              151015                                                                        ProProLeuLysLeuMetHisSerPheCysAlaPheLysAlaAspAsp                              202530                                                                        GlyProCysArgAlaIleMetLysArgPhePhePheAsnIlePheThr                              354045                                                                        ArgGlnCysGluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsn                              505560                                                                        ArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAspAsn                              65707580                                                                      AlaAsnArgIleIleLysThrThrLeuGlnGlnGluLysProAspPhe                              859095                                                                        CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg                              100105110                                                                     TyrPheTyrAsnAsnGlnThrLysGlnCysGluArgPheLysTyrGly                              115120125                                                                     GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys                              130135140                                                                     AsnIleCysGluAspGlyProAsnGlyPheGlnValAspAsnTyrGly                              145150155160                                                                  ThrGlnLeuAsnAlaValAsnAsnSerLeuThrProGlnSerThrLys                              165170175                                                                     ValProSerLeuPheGluPheHisGlyProSerTrpCysLeuThrPro                              180185190                                                                     AlaAspArgGlyLeuCysArgAlaAsnGluAsnArgPheTyrTyrAsn                              195200205                                                                     SerValIleGlyLysCysArgProPheLysTyrSerGlyCysGlyGly                              210215220                                                                     AsnGluAsnAsnPheThrSerLysGlnGluCysLeuArgAlaCysLys                              225230235240                                                                  LysGlyPheIleGlnArgIleSerLysGlyGlyLeuIleLysThrLys                              245250255                                                                     ArgLysArgLysLysGlnArgValLysIleAlaTyrGluGluIlePhe                              260265270                                                                     ValLysAsnMet                                                                  275                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LysThrLysArgLysArgLysLysGlnArgValLysIleAlaTyrGlu                              151015                                                                        GluIlePheValLysAsnMet                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      LysLysLysLysLysMetProLysLeuArgPheAlaSerArgIleArg                              151015                                                                        LysIleArgLysLysGlnPhe                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AlaLysLeuAsnCysArgLeuTyrArgLysAlaAsnLysSerSerLys                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ThrSerAspGlnIleHisPhePhePheAlaLysLeuAsnCysArg                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      SerGluLysThrLeuArgLysTrpLeuLysMetPheLysLysArgGlu                              151015                                                                        LeuGluGluTyr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      HisArgHisHisProArgGluMetLysLysArgValGluAspLeu                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      PheArgLysLeuThrHisArgLeuPheArgArgAsnPheGlyTyrThr                              151015                                                                        LeuArg                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      LeuTyrLysLysIleLeuLysLysLeuLeuGluAla                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AsnGlyLeuLysArgAspLysLeuGlyCysGluTyrCysGluCysArg                              151015                                                                        ProLysArgLysLeuIleProArgLeuSer                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 161 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AspSerGluGluAspGluGluHisThrIleIleThrAspThrGluLeu                              151015                                                                        ProProLeuLysLeuMetHisSerPheCysAlaPheLysAlaAspAsp                              202530                                                                        GlyProCysArgAlaIleMetLysArgPhePhePheAsnIlePheThr                              354045                                                                        ArgGlnCysGluGluPheIleTyrGlyGlyCysGluGlyAsnGlnAsn                              505560                                                                        ArgPheGluSerLeuGluGluCysLysLysMetCysThrArgAspAsn                              65707580                                                                      AlaAsnArgIleIleLysThrThrLeuGlnGlnGluLysProAspPhe                              859095                                                                        CysPheLeuGluGluAspProGlyIleCysArgGlyTyrIleThrArg                              100105110                                                                     TyrPheTyrAsnGlnGlnThrLysGlnCysGluArgPheLysTyrGly                              115120125                                                                     GlyCysLeuGlyAsnMetAsnAsnPheGluThrLeuGluGluCysLys                              130135140                                                                     AsnIleCysGluAspGlyProAsnGlyPheGlnValAspAsnTyrGly                              145150155160                                                                  Thr                                                                           (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="Adapter"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CCGCGGGGC9                                                                    (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GCTCCGCGGTGGCGATTCTGAGGAGGAGATGAAGAAC37                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TCTGTCGACTCACATATTTTTAACAAAAATTTCTTCAT38                                      (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="adapter"                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TCTAGATAAAAGA13                                                               (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ATCTCTAGATAAAAGAGATTCTGAGGAAGATGAAGAAC38                                      (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TCTGTCGACTCAGGTTCCATAATTATCCACCT32                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AGGTATTTTTATAACAATCAGACAAAACAGTGT33                                           (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GAAACGTTCACACTGTTTTGTCTGATTGTTATA33                                           (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CCAGCTCAATGCTGTGAATAACTCCCTGACTCCG34                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CTTGGTTGATTGCGGAGTCAGGGAGTTATTCACAGC36                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GGGGGAAATGAAAACAATTTTACTTCCAAACAA33                                           (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CCTCAGACATTCTTGTTTGGAAGTAAAATTGTTTTC36                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CCGATGCATTCATTTTGTGCATTC24                                                    (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CCTCATGATTGCCCGACATGGGCC24                                                    (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GGTCCGCGGTGGTGATGCTGCTCAGGAGC29                                               (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GCAATGTTGTTTTTTCTATCCTCCAGCAAGCAT33                                           (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GGATAGAAAAAAACAACATTGCAACAAGAAAAGC34                                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="primer"                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GGTTCTTGCATTCTTCCAGTGTCTCAAAATTG32                                            __________________________________________________________________________

We claim:
 1. A method of treating septic shock comprising administeringa chimeric protein to a mammal in need of such treatment, said chimericprotein comprising:(a) a Kunitz-type domain 1 of Tissue Factor PathwayInhibitor-2 (TFPI-2), and (b) a Kunitz-type domain 2 of Tissue FactorPathway Inhibitor (TFPI); or (c) a Kunitz-type domain 1 of TFPI, and (d)a Kunitz-type domain 2 of TFPI-2.
 2. The method of claim 1, wherein saidchimeric protein is represented by the generic structure:

    A-(X.sub.1).sub.a -B-(X.sub.2).sub.b -C

wherein A and C are independently optional flanking peptides, theflanking peptides containing 0-100 amino acids; wherein B is an optionalspacer peptide, the spacer peptide containing 0-25 amino acids; whereineach X₁ is -D-K₁ -E-where D,E are independently polypeptides of 0.25amino acids, where K₁ comprises TFPI Kunitz-type domain 1 or TFPI-2Kunitz-type domain 1; wherein each X₂ is -F-K₂ -G-where F,G areindependently polypeptides of 0-25 amino acids, where K₂ comprises TFPIKunitz-type domain 2, or TFPI-2 Kunitz-type domain 2; wherein a, b areintegers from 0-6; wherein A, B, C, D, E, F, G may comprise portions ofnative TFPI or TFPI-2 sequences or non-native sequence; andand themolecule is not native TFPI or TFPI-2.
 3. The method of claim 2, whereinA or C comprises Kunitz-type domain 3 of TFPI.
 4. The method of claim 2,wherein A or C comprises Kunitz-type domain 3 of TFPI-2.
 5. The methodof claim 2, wherein at least one of said flanking peptides comprises anamino acid sequence that binds one or more cell surface components. 6.The method of claim 5, wherein said amino acid sequence that binds oneor more cell surface components is an amino acid sequence that binds aglycosaminoglycan.
 7. The method of claim 6, wherein said amino acidsequence that binds a glycosaminoglycan is an amino acid sequence thatbinds heparin.
 8. The method of claim 7, wherein said amino acidsequence that binds heparin is a heparin-binding domain from a protein,said protein selected from the group consisting of:(a) protease nexin-1; (b) protease nexin-2; (c) antithrombin III; (d) heparin cofactor II;(e) protein C inhibitor; (f) platelet factor 4; (g) bovine pancreatictrypsin inhibitor; and (h) ghilanten-related inhibitors.
 9. The methodof claim 7, wherein said amino acid sequence that binds heparin is aheparin-binding domain selected from the group consisting of:(a) SEQ IDNO: 10; (b) SEQ ID NO: 11; (c) SEQ ID NO: 12; (d) SEQ ID NO: 13; (e) SEQID NO: 14; (f) SEQ ID NO: 15; (g) SEQ ID NO: 16; (h) SEQ ID NO: 17; and(i) SEQ ID NO:
 18. 10. The method of claim 5, wherein said flankingpeptide comprises the C-terminal tail of TFPI SEQ ID NO: 7!.
 11. Themethod of claim 5, wherein said flanking peptide comprises theC-terminal tail of TFPI-2 SEQ ID NO: 8!.
 12. The method of claim 2,wherein each K₁ is Kunitz-type domain 1 of TFPI-2, each K2 isKunitz-type domain 2 of TFPI, and a and b are integers greater than 1.13. The method of claim 1, wherein said chimeric protein is representedby the generic structure:

    A- X.sub.1 -B-X.sub.2 !.sub.n -C

wherein A and C are optional flanking peptides, the flanking peptidesindependently containing 0-100 amino acids; wherein B is an optionalspacer peptide, the spacer peptide containing 0-25 amino acids; whereineach X₁ is -D-K₁ -E-where D,E are independently peptides of 0-25 aminoacids, where K₁ is Kunitz-type domain 1 from TFPI or TFPI-2; whereineach X₂ is -F-K₂ -G-where F,G are independently peptides of 0-25 aminoacids, where K₂ is Kunitz-type domain 2 from TFPI or TFPI-2; wherein nis an integer from 1-10.
 14. The method of claim 13, wherein A or Ccomprises Kunitz-type domain 3 of TFPI SEQ ID NO: 7!.
 15. The method ofclaim 13, wherein A or C comprises Kunitz-type domain 3 of TFPI-2 SEQ IDNO: 8!.
 16. The method of claim 15, wherein said amino acid sequencethat binds glycosaminoglycan is an amino acid sequence that bindsheparin.
 17. The method of claim 16, wherein said amino acid sequencethat binds heparin is a heparin-binding domain from a protein, saidprotein selected from the group consisting of:(a) protease nexin- 1; (b)protease nexin-2; (c) antithrombin III; (d) heparin cofactor II; (e)protein C inhibitor; (f) platelet factor 4; (g) bovine pancreatictrypsin inhibitor; and (h) ghilanten-related inhibitors.